The genome sequence of Ashworth’s Rustic, Xestia ashworthii (Doubleday, 1855)

We present a genome assembly from an individual male Xestia ashworthii (Ashworth’s Rustic; Arthropoda; Insecta; Lepidoptera; Noctuidae). The genome sequence is 726.3 megabases in span. Most of the assembly is scaffolded into 31 chromosomal pseudomolecules, including the z sex chromosome. The mitochondrial genome has also been assembled and is 15.39 kilobases in length.


Background
Ashworth's Rustic, Xestia ashworthii (Doubleday, 1855) is a medium-sized (16-20 mm forewing length: Waring et al., 2017) noctuid, that Henry Doubleday based on a specimen collected at Llangollen in Denbighshire in 1853 by Joseph Ashworth.In the UK at least, the forewing ground colour is ashy to dark grey with darker grey cross markings, the post reniform one narrow and very jagged.
Ashworth's Rustic is found only in Wales in the UK, and more specifically areas of north and west Wales (NBN Atlas Partnership, 2023;Randle et al., 2019).It was found in Anglesey in 1994 (Waring et al., 2017).In Europe also, it is associated with mountainous regions and areas of exposed rock and scree slopes, ranging from the Pyrenees, Eastern Europe, Southern Scandinavia, and sparsely distributed from Italy, Greece, and eastwards in Asia to Lake Baikal (GBIF Secretariat, 2023).
In Wales there are relatively few records of Ashworth's Rustic annually due to low sampling intensity.However, where concerted efforts have been made, the species has been found to be locally abundant (Morgan, 1974).Randle et al. (2019) were unable to characterise distribution and abundance changes due to under-recording.The species was classified as nationally rare in 2019 review of the status of the macromoths of Great Britain (Fox et al., 2019) or Nationally Scarce A (Waring et al., 2017).It is also a priority species on the UK Biodiversity Action Plan (BRIG, 2007).
Biological details owe much to Tait (1923), Crewdson (1964), andMorgan (1974).Adults fly in the UK from end of June and into early August with a peak in mid-July (Randle et al., 2019).
Eggs are initially white and then turn brown over a few days, and are laid in batches on various plants, including grass stems and exposed heather roots.Heath Bedstraw (Galium saxatile) and heathers seem to be the most important foodplants (Bretherton et al., 1983: 182 characterise them as polyphagous).The looping small larvae overwinter from autumn, and can be found feeding again from February onwards, with large caterpillars, 4 cm fully fed, grey, with a large wedge-shaped black marking bilaterally on each segment, often found in April and May apparently basking on exposed rocks or near the top of vegetation.Pupae have been recovered from cracks and crevices in rocks, and from shallow chambers under the soil, where they form a flimsy cocoon (Bretherton et al., 1983: 182).
Tait (1923) reported that the previously easily-accessible and apparently healthy populations around Penmaenmawr and the Sychnant Pass suffered greatly from attack by a still unidentified ichneumonid wasp around 1917-18.A nationally notable tachinid fly, Drino lota (Meigen, 1824) has been reported as a parasitoid of X. ashworthii (Smith, 1989: 124).
The recent finding that cool-adapted moths in Great Britain are increasingly shifting their range to the north-west (Hordley et al., 2023) raises significant issues for Ashworth's Rustics, as there is limited habitat available to expand into in north Wales before they meet the Irish Sea, and even elevational shifts as seen in mountain butterflies (Rödder et al., 2021) can only occur for so long before they reach the peak.This genome sequence will not only be an invaluable resource for determination of levels of genetic differentiation within and between the subspecies, and their relationships, also to other Xestia, but will be of great help in developing genetic markers to assess levels of genetic diversity and patterns of gene flow within the Welsh and continental populations, and for detecting differential patterns of selection and evolutionary trajectories across their range.

Genome sequence report
The genome was sequenced from one male Xestia ashworthii (Figure 1) collected from Llanfihangel-y-Pennant, Wales (52.67,.A total of 29-fold coverage in Pacific Biosciences single-molecule HiFi long reads was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 5 missing joins or mis-joins and removed 7 haplotypic duplications, reducing the assembly length by 0.42% and the scaffold number by 6.67%. The final assembly has a total length of 726.3 Mb in 69 sequence scaffolds with a scaffold N50 of 25.1 Mb (Table 1).The snailplot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC  proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (99.55%) of the assembly sequence was assigned to 31 chromosomal-level scaffolds, representing 30 autosomes and the Z sex chromosome.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial genome was also assembled and can be found as a contig within the multifasta file of the genome submission.

Sample acquisition and nucleic acid extraction
A male Xestia ashworthii (specimen ID NHMUK014425722, ToLID ilXesAshw1) was collected from Llanfihangel-y-Pennant, Wales, UK (latitude 52.67, longitude -3.96) on 2021-08-01, using a light trap.The specimen was collected and identified by David Lees (Natural History Museum) and preserved by dry freezing at -80 °C.

Assembly accession
A Hi-C map for the final assembly was produced using bwa-mem2 (Vasimuddin et al., 2019) in the Cooler file format (Abdennur & Mirny, 2020).To assess the assembly metrics, the k-mer completeness and QV consensus quality values were calculated in Merqury (Rhie et al., 2020).This work was done using Nextflow (Di Tommaso et al., 2017) DSL2  agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.
Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material

Software tool Version
Is the rationale for creating the dataset(s) clearly described?Yes

Are the protocols appropriate and is the work technically sound? Yes
Are sufficient details of methods and materials provided to allow replication by others?Yes

Are the datasets clearly presented in a useable and accessible format? Yes
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Compartive genomics, population genomics, conservation I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Comments on the manuscript:
The first sentence of background can be modified as "Ashworth's Rustic Xestia ashworthii (Doubleday, 1855) is a medium-sized (wingspan 16-20 mm) noctuid moth (Waring et al., 2017)"."that Henry Doubleday based on a specimen………………….." can be removed.Not necessary to give the details here.

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The sentence can be modified as "The species was also observed in Anglesey in 1994 (Waring et al., 2017)".

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Common name of the species may not be used instead of scientific name.Authors have used common name of the species in many places.The species name can be included in the first sentence of the third paragraph."In Wales there are relatively few records of X. ashworthii annually……………………………….."

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Fifth paragraph second sentence within the parenthesis "182 characterise them as polyphagous".Is it necessary to give here?Otherwise, details of the polyphagous can be given in separate sentence elaborately.There is little confusion here.

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In the genome sequence report the first sentence can be modified as "The genome was sequenced from a male X. ashworthii……"

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In the Methods, First paragraph first sentence can be rewritten as "The genus name can be given in short form like X. ashworthii……………………" ○ Protein-coding genes and gene transcripts details aren't given in the article.

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Authors have used standard laboratory protocol for DNA isolation.Software and techniques were for the annotations and assembly of the sequences.
○ Above all, I confirm that the manuscript meets the necessary scientific standard and is suitable for indexing.
Is the rationale for creating the dataset(s) clearly described?

Axel Künstner
University of Lübeck, Lübeck, Germany The authors present the genome assembly of Xestia ashworthii (Ashworth's Rustic), a medium-sized noctuid with ZW sex chromosomes.The manuscript is well written and focuses on the genome assembly of the homogametic sex (male).The reported total length of the assembled genome is 726.3Mb in 69 scaffolds, which were assigned to 30 autosomes and the Z chromosome.
The methods used are sound, software resources used are well described, and the data is available via the European Nucleotide Archive.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes

Are sufficient details of methods and materials provided to allow replication by others? Yes
Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Cancer genomics, Microbiome, Bioinformatics, Computational Biology I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.Reviewer Expertise: Population genomics, genome assembly and annotation I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Figure 2 .
Figure 2. Genome assembly of Xestia ashworthii, ilXesAshw1.1:metrics.The BlobToolKit Snailplot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 726,328,142 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (42,390,235 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (25,131,726 and 16,416,265 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the lepidoptera_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/Xestia%20ashworthii/dataset/ilXesAshw1_1/snail.

Figure 5 .
Figure 5. Genome assembly of Xestia ashworthii, ilXesAshw1.1:Hi-C contact map of the ilXesAshw1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=LKUwmbHETYaIrDZ4kLlArw.

Reviewer Report 11
May 2024 https://doi.org/10.21956/wellcomeopenres.22692.r75003© 2024 Paris J.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Josephine Paris Department of Health, Life and Environmental Sciences, University of L'Aquila, Coppito, Italy In this report, the assembled genome of Xestia ashworthii is presented.The Introduction provides a nice and very detailed background on the species and its distribution.I only stumbled upon one part, which reads, "Heath Bedstraw (Galium saxatile) and heathers seem to be the most important foodplants (Bretherton et al., 1983: 182 characterise them as polyphagous" It would be nice to know what the 182 refers to, as currently it is unclear to me.The assembly was performed to a good standard using Pacbio Hifi data and scaffolded with Hi-C data.Assembly contiguity and completeness are well demonstrated.It would be nice to include the amount (in Gb) of Pacbio Hifi (not only the coverage) and Hi-C data that was generated.The datasets are clearly available via the links provided.Is the rationale for creating the dataset(s) clearly described?Yes Are the protocols appropriate and is the work technically sound?Yes Are sufficient details of methods and materials provided to allow replication by others?Yes Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.

Table 3 . Software tools: versions and sources.
Reviewer Report 15 August 2024 https://doi.org/10.21956/wellcomeopenres.22692.r91594This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Partly Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
https://doi.org/10.21956/wellcomeopenres.22692.r91598© 2024 Künstner A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.